ABSTRACT Liver transplantation currently represents the only treatment for end stage liver disease (ESLD), the 8th most frequent cause of death in the United States (US). Efforts to overcome the chronic shortage of transplantable human livers include attempts at alternative organ procurement and replacement strategies. The most promising approach to date that provides the appropriate matrix composition and supporting vascular structures has been the development of perfusion decellularization, which enables the removal of cellular material for a native organ while maintaining the native matrix, structure and vascular of the liver. Utilizing perfusion decellularization and recellularization technology, several groups have demonstrated the ability to seed a variety of liver-specific cell types into decellularized liver constructs, however reconstitution of the endothelial cell lining of the vascular networks in these scaffolds has remained a significant challenge to the development of a therapeutic bioengineered liver (BEL). Miromatrix Medical Inc. has recently reported revascularizing a clinically translatable porcine-derived liver scaffold and demonstrated in vivo graft patency in a large animal model without sustained administration of anticoagulant therapies. Solving the critical vascular constraint allows this Direct to Phase II SBIR project to focus on production and testing of a fully functional BEL to address the chronic shortage of transplantable livers and develop new therapeutic options to those with ESLD utilizing Miromatrix? broad proprietary perfusion decellularization and recellularization technology. In Specific Aim #1, we will demonstrate recellularized liver graft function in an acute liver failure model. The optimal media and seeding conditions for hepatocyte functionality will be selected (1.1). Equivalency of human hepatocytes to porcine hepatocytes will be demonstrated (1.2), and vascular patency of endothelial and hepatocyte recellularized livers will be confirmed in acute blood loops (1.3). Forty-eight hour functionality of this liver graft will be demonstrated in pigs (1.4). In Specific Aim #2, we will optimize the seeding of cholangiocytes into liver grafts recellularized with endothelial cells and hepatocytes and identify assays that will allow assessment of cholangiocyte function after seeding to verify successful engraftment (2.1). We will then create tri-culture grafts using only human cells and test for equivalency to the porcine+human grafts (2.2) and determine whether addition of cholangiocytes to endothelial+hepatocyte liver grafts adversely affects graft patency (2.3). Finally, in Specific Aim #3, we will demonstrate the BEL functionality in an orthotopic chronic liver failure model by developing a transplant model and evaluating baseline data (3.1) and by demonstrating liver functionality for fourteen days in a recovery model in pigs (3.2). Data generated will be used in a Pre-IND submission meeting to define the testing required for complete human recellularization and functional testing of the BEL grafts to initiate clinical studies.